The overall goal of this K18 proposal is to develop a program for the study of DNA replication during the differentiation of mouse (mES) and human (hES) embryonic stem (ES) cells. The candidate has made significant contributions to our understanding of how DNA replication is regulated during the cell-cycle and would now like to apply this expertise to the study of replication during early mammalian development. Changes in origin specification and replication timing have been observed between established and/or differentiated cell lines. These changes coincide with large scale chromatin remodeling and likely take place early in development during major cell fate decisions. We will develop both mES and hES cell systems, mES cells offer sophisticated genetic tools and differentiation systems. One objective will be to perform genome-wide replication timing and gene expression analysis at different stages during the differentiation of mES cells, and to examine the effects of specific mutations on the regulation of the replication program. By contrast, I hES cells have the advantage of a nearly completely sequenced genome, allowing for the construction of complete coverage DNA arrays that are not yet available for mice. Since replication origins in mammals are not spec fed by obvious DNA sequences, and since they are largely excluded from transcription units, their unbiased identification requires complete-coverage genomic arrays. Hence, a second objective is to identify the positions of replication origins along the length of human chromosome 22, in collaboration with Mike Snyder at the Yale Genome Analysis center. Particularly interesting to us is the potential to identify origins that are regulated during development, which will require homogeneous differentiation systems for hES cells paralleling those recently developed in mouse. The hES cells are referenced by SLA#03-W134, WiCell Ref P98222US and as cell lines WA01 and WA09. This award would coincide with a sabbatical, which will begin with a 2-3 week visit to Geron, Co. (Menlo Park, CA) to learn their latest hES differentiation techniques, followed by work in the home Labto map replication origins. A second phase will involve a 2-3 month period with Peter and Joy Rathjen (U. of Adelaide), who have developed systems to differentiate mES cells homogeneously through defined intermediates. Homogeneous differentiation will allow us to identify the precise cellcycle during which specific replication timing changes take place. Conditions for homogeneous differentiation of mES cells will also be applied to hES cells in work aimed at the production of hES cell differentiation intermediates for replication studies.